Immunohistochemical analysis of MCT1, MCT2 and MCT4 expression in rat plantaris muscle

Abstract

We addressed the need for histological assessment of myocellular domains occupied by monocarboxylate transporters (MCT1, MCT2 and MCT4). From the perspective of lactate shuttle hypotheses we posited that MCT1 would be highly expressed in oxidative fibres, whereas MCT4 would be found in highly glycolytic fibres. Furthermore, we hypothesized that MCT1 would be detected at interfibrillar as well as at subsarcolemmal and sarcolemmal cell domains, whereas MCT2 and MCT4 abundances would be most prominent at the sarcolemma. To test these hypotheses, we examined cellular locations of MCT1, MCT2 and MCT4 transporter proteins in different fibre types (slow oxidative, SO; fast oxidative glycolytic, FOG; fast glycolytic, FG) in rat plantaris muscles by the avidin-biotin complex (ABC) as well as other methods. The plantaris was used as it is a mixed fibre skeletal muscle. MCTs, glucose transporter (GLUT4) protein, and mitochondrial constituent cytochrome oxidase (COX) abundances were assessed by immunohistochemistry and Western blotting using affinity-purified antibodies. The staining method was specific and stable, which allowed for semiquantitative assessment of MCT expression. As well, confocal laser scanning microscopy assessed MCT isoform localizations. Findings of the present study were: (1) MCT1 is located at the sarcolemma and throughout the cell interior in SO and FOG fibres where the mitochondrial reticulum was present; (2) in contrast, MCT4 was highly expressed in the sarcolemmal domain of FG and FOG fibres but poorly expressed in SO fibres; and (3) confocal laser-scanning microscopy demonstrated that MCT1 and COX are co-localised at both interfibrillar and subsarcolemmal cell domains, whereas MCT2 is only faintly detected at the sarcolemma of oxidative fibres. MCTs and associated proteins are positioned to facilitate the function of the lactate shuttles.

<b>Cellular distributions of monocarboxylate transporters: a r</b><b>eview </b>

“…Another type of transporter for monocarboxylates, SLC5A8, was identified in 2003 (87). Since its transport via SLC5A8 is coupled with Na + (19,96), it has been termed a Sodium-dependent MonoCarboxylate the skeletal muscle, Hashimoto et al (60) have ascribed the results of unstaining to the fixation itself: immunoreactivities of MCT1 and MCT4 are blocked in the formalin-fixed sections in contrast to unfixed sections. However, the different or unsuccessful stainability may be more likely due to antibody specificity, animal species, or types of sections.…”

“…However, most immunohistochemical studies have failed to demonstrate MCTs in mitochondria at the electron microscopic level (71) (also our unpublished data). At the light microscopic level, MCT1 signals have overlapped with a mitochondrial inner membrane protein (COX) in the rat skeletal muscle (60). The transport of monocarboxylates, mainly pyruvate, may be mediated by another proton-linked carrier known as the Mitochondrial Pyruvate Carrier (MPC) (54).…”

Evidence for a stromal-epithelial “lactate shuttle” in human tumors

“…3,11 In contrast, MCT1/2 transporters facilitate the uptake of lactate, consistent with their expression in slow-twitch muscle fibers (MCT1) and neurons (MCT2). 3,12 In the present study, we set out to determine if a similar compartmentalization of lactate transporters occurs in a subset of human breast cancers, in which cancer-associated fibroblasts undergo aerobic glycolysis. This subset of breast cancers is defined by a loss of stromal Cav-1, 13 which appears to be an indicator of hypoxia, oxidative stress and autophagy in the tumor stroma and creates a "lethal" tumor microenvironment.…”

“…photoreceptor cells, and in skeletal muscle by the fast-twitch and slow-twitch fibers. [1][2][3][4][5][6][7]12,50,51 In normal tissue, such as skeletal muscle and brain, it has been found that glycolytic and oxidative cells are metabolically-coupled, via monocarboxylate transporters (MCTs). Under aerobic conditions, fast-twich muscle fibers and astrocytes metabolize glucose through aerobic glycolysis, producing lactate that is extruded via MCT4.…”

Nonenzymatic Proton Handling by Carbonic Anhydrase II during H+-Lactate Cotransport via Monocarboxylate Transporter 1

“…In the brain MCT1 is located mainly in glial cells, where it facilitates the export of lactate, which is then taken up by neurons via the high affinity MCT2; thereby the two isoforms are believed to shuttle lactate from glial cells to neurons which seems to be pivotal for brain energy metabolism (3)(4)(5)(6). In muscle, MCT1 is highly expressed in oxidative type I fibers, where it mediates import of lactate that is released by glycolytic muscle cells via MCT3 and MCT4 (7)(8)(9). MCT1, when expressed in Xenopus oocytes, operates with a K m value of 3.5 mM for lactate influx and of 6.4 mM for efflux of lactate.…”